Conventionally, a technique for degrading a sugar chain by a glycosidase to derivatize the sugar chain has been utilized in the analytical studies on a several milligram scale such as structural analysis of a sugar chain. However, since derivatives of individual sugar chains could not be obtained in a large amount, development of the studies on a gram scale has been retarded. Therefore, it has been difficult to apply a sugar chain derivative in the synthesis studies such as the manufacture of pharmaceuticals.
On the other hand, it has been known that a glycopeptide is obtained in a large amount from an egg yolk (Biochimica et Biophysica Acta 1335 (1997) p 23–32). However, there has not been reported a case where a fluorenylmethoxycarbonylated (Fmoc-)sugar chain derivative of a compound 1 shown in FIG. 1, or a series of compounds having deletions of several sugar residues such as sialic acid or galactose on one of non-reducing terminals of a branched sugar chain in the compound 1, are obtained in a large amount. In addition, there are some cases where several sugar chains are isolated in a small amount from a protein or the like in human blood. However, when the sugar chain is employed in the manufacture of a pharmaceutical, there is a risk of allowing the pharmaceutical to contaminate AIDS virus, hepatitis viruses or the like. Therefore, there has been a controversy over the technique of applying the sugar chain to a pharmaceutical.
Meanwhile, there are numerous examples of processes for preparing sugar chains of which branched moieties have the same structure for a branched sugar chain. As conventional techniques, there are three kinds of processes. A first is a process for isolating and purifying an asparagine type sugar chain complex from a naturally occurring glycoprotein. Representative examples of this type are those reported in T. Tamura, et al., Anal. Biochem., 1994, 216, p 335–344, V. H. Thomas, et al., Carbohydr. Res., 1998, 306, p 387–400, K. G. Rice, et al., Biochemistry, 1993, 32, p 7264–7270 and the like. The advantage of these processes is that the synthesis of the sugar chain is not necessary. However, there are several defects. For instance, the sugar chain derived from the above glycoprotein is obtained as a mixture of the sugar chains having random deletions of several sugar residues in non-reducing terminal moieties, and the sugar chains contained in the above mixture are similar to each other in their physical and chemical characteristics, so that it is very difficult to separate into individual sugar chains, thereby making it substantially impossible to obtain a single sugar chain in a large amount. Also, there is a case where a sugar chain has been isolated from a protein in human blood (isolation from Fibrinogen: C. H. Hokke, et al., Carbohydr. Res., 305 (1997), p 463–468, isolation from Human Serum Transferrin: M. Mizuno et. al., J. Am. Chem. Soc., 1999, 121, p 284–290) in order to obtain the sugar chain in a relatively large amount. As mentioned above, a protein in human blood must be handled carefully because the protein may be contaminated with an AIDS virus or hepatitis viruses. Therefore, it is difficult to utilize the resulting sugar chains and derivatives thereof in the development of pharmaceuticals. Even if the sugar chains were obtained in a large amount, their structures are limited, and any cases where sugar chains and derivatives thereof having many kinds of structures are obtained do not substantially exist.
In K. G. Rice, et al., Biochemistry, 1993, 32, p 7264–7270, or Rice, et al., Neoglycoconjugate, Academic Press, 1994, ISBN 0-12-440585-1 p 286–321, a sugar residue is removed from a non-reducing terminal of a sugar chain with a glycosidase. However, since a sugar chain having a single structure used as a raw material cannot be obtained in a large amount, the process can only be carried out on an analytical scale. E. Meinjohanns (J. Chem. Soc. Perkin Trans 1, 1998, p 549–560) et al. have obtained a compound 56 shown in FIG. 5 from a bovine fetuin (bovine-derived glycoprotein) and then synthesized a compound 10 shown in FIG. 1 via a compound 33 shown in FIG. 3. In order to obtain the compound 56, which is a first raw material, a hydrazine degradation reaction has been utilized. This hydrazine is highly toxic, so that there is a problem in safety when the resulting sugar chain derivative is applied to a pharmaceutical, due to the contamination of a trace amount of the hydrazine. In addition, the sugar chain derivatives of the compounds 56, 33 and 10 to which sialic acid is not bound can be obtained only in a small amount.
A second process is a process of synthesizing a sugar chain chemically. Currently, a construction of about 10 sugars prepared by combining monosaccharides according to a chemical synthesis process can be made as shown in a reported case of J. Seifert et al., Angew Chem Int. Ed. 2000, 39, p 531–534. The advantage of this process is that all of the sugar chain derivatives can be theoretically obtained. However, since its preparation steps are enormous, there is a defect that a synthesis in a large amount is difficult. In addition, even in case where a sugar chain in which about 10 sugar residues are bound is synthesized in an amount of several milligrams, a time period of as long as about one year is required. While there have been so far some cases where several sugar chains are synthesized chemically, most of the cases actually could synthesize the intended sugar chain only in an amount as small as several milligrams.
A third process is a process of synthesizing a sugar chain by combining an enzymatic reaction and a chemical reaction. As a representative example, there is a process as reported by Carlo Unverzagt, Angew Chem Int. Ed. 1996, 35, p 2350–2353. This process employs a technique in which a sugar chain is constructed to a certain length by chemical synthesis, and thereafter a sugar residue is added to the sugar chain by an enzymatic reaction, thereby extending the sugar chain. However, since the enzyme used in the chain extension has substrate specificity, the kinds of sugar which can be introduced into the sugar chain are limited. In addition, since the preparation steps are enormous in the chemical synthesis, a large-scale synthesis is difficult, so that the final product can be obtained only in a small amount. Alternatively, in C. H. Lin et al. (Bioorganic & Medicinal Chemistry, 1995, p 1625–1630), a sialyloligoglycopeptide is obtained from an egg yolk by employing a process reported by M. Koketsu et al. (J. Carbohydrate Chemistry, 1995, 14(6), p 833–841), and the structure of the non-reducing terminal moieties of the sugar chain is modified with a glycosidase and a sugar transferase. A sugar chain having only one asparagine (Asn) residue bound to its non-reducing terminal moiety is shown in a drawing of this article. However, according to the process reported in J. Carbohydrate Chemistry, 1995, 14(6), p 833–841, a mixture of sugar chains each having on average about 2.5 amino acids other than asparagine, such as lysine, which are bound to a non-reducing terminal of the sugar chain. Therefore, a sugar chain derivative cannot be obtained as a single compound. Also, there is not suggested obtainment of individual derivatives of the compounds having arbitrary deletions of the sugar residues in the branched sugar chain of the branching sugar chains in a large amount. An article by C. H. Lin et al. does not give any evidence that a sugar chain is obtained as a single product. Y. Ichikawa mentions in Glycopeptide and Related Compounds (Marcel Dekker, Inc., 1997, ISBN 0-8247-9531-8, p. 79–205) that if the trifurcated sugar chain complex is sequentially treated from a terminal with a glycosidase, sugar chains can be sequentially removed from a non-reducing terminal of the sugar chain, thereby giving various sugar chain derivatives. However, how the individual sugar chains are separated after the enzymatic treatment is not described, and the synthesis is limited only to those having uniform branching. Therefore, even with this process, it is thought that the individual derivatives of the compounds having arbitrary deletions of sugar residues in the branched sugar chain of the branching sugar chains cannot be obtained in a large amount.